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Abstract:

The present invention provides methods for the quantification of an
unknown bioagent in a sample by amplification of nucleic acid of the
bioagent, and concurrent amplification of a known quantity of a
calibration polynucleotide from which are obtained a bioagent identifying
amplicon and a calibration amplicon. Upon molecular mass analysis, mass
and abundance data are obtained. The identity of the bioagent is then
determined from the molecular mass of the bioagent identifying amplicon
and the quantity of the identified bioagent in the sample is determined
from the abundance data of the bioagent identifying amplicon and the
abundance data of the calibration amplicon.

Claims:

1. A system, comprising: a) a nucleic acid amplification component,
comprising a pair of primers designed to produce a bioagent identifying
amplicon from a bioagent nucleic acid sequence acid under amplification
conditions, a known quantity of a calibration polynucleotide comprising a
calibration sequence designed to produce a calibration amplicon as a
result of amplification with said primers under said amplification
conditions wherein the 5' and 3' ends of said amplicons are the sequences
of said pair of primers or complements thereof, and an amplification
reaction vessel wherein said bioagent identifying amplicon and said
calibration amplicon are concurrently amplified; b) a molecular mass
determination component; and c) a molecular mass identification component
comprising at least one computer comprising a processor and software, and
a database of molecular masses of bioagent identifying amplicons from a
plurality known bioagents wherein a match between the molecular mass of
said bioagent identifying amplicon and the molecular mass of a bioagent
identifying amplicon from a known bioagent in said database of molecular
masses identifies said bioagent, a molecular mass of said calibration
amplicon identifies said calibration amplicon, and molecular mass
abundance data of said bioagent identifying ampicon and molecular mass
abundance data of said calibration amplicon indicates the quantity of
said bioagent.

2. The system of claim 1, wherein said calibration sequence: a) comprises
a chosen standard sequence of a bioagent identifying amplicon with the
exception of a deletion of about 2 to about 8 consecutive nucleotide
residues of said standard sequence; b) comprises a chosen standard
sequence of a bioagent identifying amplicon with the exception of an
insertion of about 2 to about 8 consecutive nucleotide residues of said
standard sequence; or c) has at least 80% sequence identity with a chosen
standard sequence of a bioagent identifying amplicon.

3. The system of claim 1, wherein said molecular mass determination
component comprises a mass spectrometer.

4. The system of claim 1, wherein said molecular mass abundance data of
said calibration amplicon comprises a standard curve wherein the amount
of said calibration polynucleotide in said amplification reaction vessel
is varied.

5. The system of claim 1, wherein said nucleic acid amplification
component comprises a plurality of primer pairs which amplify a
corresponding plurality of bioagent nucleic acid sequences and
calibration sequences.

9. The system of claim 1, further comprising a nucleic acid purification
component.

10. The system of claim 9, wherein said nucleic acid purification
component comprises one or more buffer manipulations, one or more salt
manipulations, one or more thermal manipulations, one or more pH
manipulations, one or more mechanical manipulations, one or more
centrifugation manipulations, or one or more magnetic manipulations.

11. The system of claim 1, wherein said nucleic acid amplification
component comprises a thermocycler.

12. The system of claim 1, wherein said nucleic acid amplification
component comprises one or more salts, one or more buffers, one or more
purified oligonucleotide primers, one or more dNTPs, or one or more
enzymes.

13. The system of claim 1, further comprising a computer program on a
computer readable medium configured to direct said processor to
coordinate the operation of said nucleic acid amplification component,
said molecular mass determination component, and said molecular mass
identification component.

14. A system, comprising: a) a nucleic acid amplification component,
comprising a pair of primers designed to produce a bioagent identifying
amplicon from a bioagent nucleic acid sequence acid under amplification
conditions, a known quantity of a calibration polynucleotide comprising a
calibration sequence designed to produce a calibration amplicon as a
result of amplification with said primers under said amplification
conditions wherein the 5' and 3' ends of said amplicons are the sequences
of said pair of primers or complements thereof, and an amplification
reaction vessel wherein said bioagent identifying amplicon and said
calibration amplicon are concurrently amplified; b) a base composition
determination component comprising a component that measures the
molecular masses of said bioagent identifying amplicon and said
calibration amplicon, and a computer comprising a processor and software
that calculates the base compositions of said molecular masses; and c) a
base composition identification component comprising at least one
computer comprising a processor and software, and a database of base
compositions of bioagent identifying amplions from a plurality of known
bioagents wherein a match between the base composition of said bioagent
identifying amplicon and the base composition of a bioagent identifying
amplicon from a known bioagent in said database of base compositions
identifies said bioagent, a base composition of said calibration amplicon
identifies said calibration amplicon, and molecular mass abundance data
of said bioagent identifying ampicon and molecular mass abundance data of
said calibration amplicon indicates the quantity of said bioagent.

15. The system of claim 14, wherein said calibration sequence: a)
comprises a chosen standard sequence of a bioagent identifying amplicon
with the exception of a deletion of about 2 to about 8 consecutive
nucleotide residues of said standard sequence; b) comprises a chosen
standard sequence of a bioagent identifying amplicon with the exception
of an insertion of about 2 to about 8 consecutive nucleotide residues of
said standard sequence; or c) has at least 80% sequence identity with a
chosen standard sequence of a bioagent identifying amplicon.

16. The system of claim 14, wherein said molecular mass determination
component comprises a mass spectrometer.

17. The system of claim 14, wherein said molecular mass abundance data of
said calibration amplicon comprises a standard curve wherein the amount
of said calibration polynucleotide in said amplification reaction vessel
is varied.

18. The system of claim 14, wherein said nucleic acid amplification
component comprises a plurality of primer pairs which amplify a
corresponding plurality of bioagent nucleic acid sequences and
calibration sequences.

[0004] Information about the identity and total amount of microbes in
biological samples is of prime importance in medicine in order to assess
the risk of infectious disease, to diagnose infections and predict their
clinical course. In a variety of other areas such as food product
monitoring, bioremediation, microbial forensics and biowarfare/bioterror
investigations, efficient and cost effective methods for quantification
of microbial bioagents are needed. In addition, determination of the
quantity of a bioagent (microbe, bacterium, virus, fungus, etc.) is a
common endeavor in microbiology in the fields of clinical diagnostics,
epidemiology, forensics, bioremediation, and quality control.

[0005] Methods currently in use for detection and determination of
bacteria include bacterial culture and microscopy, detection of bacterial
metabolites, and identification of surface molecules by specific
antibodies.

[0006] The polymerase chain reaction (PCR) is only a qualitative method
due to its exponential time course and equally exponential amplification
of errors. Efforts have been made to convert PCR to a quantitative
method. Among the variety of quantitative PCR methods, are methods
depending upon external standardization and on internal standardization.
Among the latter, competitive PCR methods are based on co-amplification
of a target DNA with a standard competitor DNA which competes with the
template DNA for the same set of amplification primers. Since the
competitor is added to the PCR reaction mixture in known amounts, it is
possible to calculate the amount of target DNA from the experimental
determination of the ratio of amplified products of sample and standard
competitor DNA.

[0007] Methods for rapid and cost effective identification of microbial
bioagents through molecular mass measurement of amplification products by
molecular mass analysis of bioagent identifying amplicons are disclosed
and claimed in U.S. application Ser. Nos. 09/798,007, 09/891,793,
10/660,997, 10/660,122, 10/660,996, 10/418,514 and 10/728,486, each of
which is commonly owned and incorporated herein by reference in its
entirety. These methods and others would derive great benefit from a
means of determination of the quantity of any given microbial bioagent
present in a biological sample. Quantification of organisms can be very
valuable, particularly in a clinical setting, like Hepatitis C for
example, where the greater the number of infectious organisms generally
correlates with a less healthy patient and a more difficult clinical
course.

[0008] The methods described herein satisfy the need for methods for
concurrent identification and quantification of bioagents, as well as
other needs, by providing internal calibration using a nucleic acid
standard calibrant in an amplification reaction.

SUMMARY OF THE INVENTION

[0009] The present invention provides methods for determination of the
quantity of an unknown bioagent in a sample by contacting the sample with
a pair of primers and a known quantity of a calibration polynucleotide
that comprises a calibration sequence. Nucleic acid from the bioagent in
the sample is concurrently amplified with the pair of primers and
amplifying nucleic acid from the calibration polynucleotide in the sample
with the pair of primers to obtain a first amplification product
comprising a bioagent identifying amplicon and a second amplification
product comprising a calibration amplicon. The sample is then subjected
to molecular mass analysis resulting in molecular mass and abundance data
for the bioagent identifying amplicon and the calibration amplicon. The
bioagent identifying amplicon is distinguished from the calibration
amplicon based on molecular mass wherein the molecular mass of the
bioagent identifying amplicon provides a means for identifying the
bioagent. Comparison of bioagent identifying amplicon abundance data and
calibration amplicon abundance data indicates the quantity of bioagent in
the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 shows a representative process diagram for identification
and determination of the quantity of a bioagent in a sample.

[0011] FIG. 2 shows a representative mass spectrum of a viral bioagent
identifying amplicon for the RdRp primer set of the SARS coronavirus
(SARS) and the corresponding RdRp calibration amplicon.

[0013] The figures depict preferred embodiments of the present invention
for purposes of illustration only. One skilled in the art will readily
recognize from the following discussion that alternative embodiments of
the structures and methods illustrated herein may be employed without
departing from the principles of the invention described herein.

DESCRIPTION OF EMBODIMENTS

[0014] The present invention provides methods for identification and
determination of the quantity of a bioagent in a sample. Referring to
FIG. 1, to a sample containing nucleic acid of an unknown bioagent are
added primers (100) and a known quantity of a calibration polynucleotide
(105). The total nucleic acid in the sample is then subjected to an
amplification reaction (110) to obtain amplification products. The
molecular masses of amplification products are determined (115) from
which are obtained molecular mass and abundance data. The molecular mass
of the bioagent identifying amplicon (120) provides the means for its
identification (125) and the molecular mass of the calibration amplicon
obtained from the calibration polynucleotide (130) provides the means for
its identification (135). The abundance data of the bioagent identifying
amplicon is recorded (140) and the abundance data for the calibration
data is recorded (145), both of which are used in a calculation (150)
which determines the quantity of unknown bioagent in the sample. Each of
these features is described below in greater detail.

[0015] In one embodiment, a sample comprising an unknown bioagent is
contacted with a pair of primers which can amplify nucleic acid from the
bioagent, and a known quantity of a polynucleotide that comprises a
calibration sequence. The nucleic acids of the bioagent and of the
calibration sequence are amplified. The rate of amplification is
reasonably assumed to be similar for the nucleic acid of the bioagent and
of the calibration sequence. The amplification reaction produces two
amplification products: a bioagent identifying amplicon and a calibration
amplicon. The amplified sample containing the bioagent identifying
amplicon and the calibration amplicon is then subjected to molecular mass
analysis by mass spectrometry, for example. The resulting molecular mass
analysis of the nucleic acid of the bioagent and of the calibration
sequence provides molecular mass data and abundance data for the nucleic
acid of the bioagent and of the calibration sequence. The molecular mass
data obtained for the nucleic acid of the bioagent enables identification
of the unknown bioagent and the abundance data enables calculation of the
quantity of the bioagent, based on the knowledge of the quantity of
calibration polynucleotide contacted with the sample. The calculations
are well within the scope of those of the ordinary artisan.

[0016] A calibration sequence is a sequence chosen to represent a portion
of a genome of a bioagent (bacterium, virus etc.) that can be amplified
by a particular primer pair to yield an amplification product
(calibration amplicon) that can be distinguished on the basis of its
molecular mass from an analogous amplification product (bioagent
identifying amplicon) obtained by amplification of native DNA of a
bioagent (bacterium, virus, etc) with the same pair of primers. One means
of distinguishing an amplification product of a calibration sequence vs.
a bioagent identifying amplicon is to design the calibration sequence so
that, upon amplification, it gives rise to an amplification product
consisting of a calibration amplicon that has a molecular mass
distinguishable from the analogous bioagent identifying amplicon. This is
desired because, as in any internally calibrated method, the calibration
sequence and the bioagent sequence are amplified concurrently in the same
amplification reaction vessel.

[0017] In some embodiments, construction of a standard curve where the
amount of calibration polynucleotide spiked into the sample is varied,
provides additional resolution and improved confidence for the
determination of the quantity of bioagent in the sample. The use of
standard curves for analytical determination of molecular quantities is
well known to one with ordinary skill and can be performed without undue
experimentation.

[0018] In some embodiments, multiplex amplification is performed where
multiple bioagent identifying amplicons are amplified with multiple
intelligent primer pairs which also amplify the corresponding standard
calibration sequences. In this or other embodiments, the standard
calibration sequences are optionally included within a single vector such
as a plasmid which functions as the calibration polynucleotide. Multiplex
amplification methods are well known to those with ordinary skill and can
be performed without undue experimentation.

[0019] In some embodiments, the calibrant polynucleotide is used as an
internal positive control to confirm that amplification conditions and
subsequent analysis steps are successful in producing a measurable
amplicon. Even in the absence of copies of the genome of a bioagent, the
calibration polynucleotide can give rise to a calibration amplicon.
Failure to produce a measurable calibration amplicon indicates a failure
of amplification or subsequent analysis step such as amplicon
purification or molecular mass determination.

[0020] In some embodiments, the calibration sequence is inserted into a
vector which then itself functions as the calibration polynucleotide. In
some embodiments, more than one calibration sequence is inserted into the
vector that functions as the calibration polynucleotide. The process of
inserting polynucleotides into vectors is routine to those skilled in the
art and can be accomplished without undue experimentation. Thus, it
should be recognized that the present invention should not be limited to
the embodiments described herein. The present invention can be applied
for determination of the quantity of any bioagent identifying amplicon
when an appropriate standard calibrant polynucleotide sequence is
designed and used. The process of choosing an appropriate vector such as
a plasmid for insertion of a calibrant is also a routine operation that
can be accomplished by one with ordinary skill without undue
experimentation.

[0021] In some embodiments of the present invention, determination of the
molecular masses of the bioagent identifying amplicon and the calibration
amplicon is accomplished using mass spectrometry. Exemplary techniques of
mass spectrometry include, but are not limited to, electrospray
ionization Fourier transform ion cyclotron resonance mass spectrometry
(ESI-FTICR-MS) and electrospray ionization time-of-flight mass
spectrometry (ESI-TOF-MS).

[0025] Calibration sequences can be routinely designed without undue
experimentation by choosing a reference sequence representing any
bioagent identifying amplicon that can be amplified by a specific pair of
primers of any class e.g: broad range survey, division-wide, clade level,
or drill down or any arbitrarily named class of primer and by deleting or
inserting about 2-8 consecutive nucleobases into that sequence such that
the calibration sequence is distinguishable by molecular mass from the
reference sequence upon which the calibration sequence is based. One will
recognize that this range comprises insertions or deletions of 2, 3, 4,
5, 6, 7, or 8 nucleobases. In other embodiments, the total insertion or
deletion of consecutive nucleobases may also exceed 8 nucleobases. In
other embodiments, the total insertion or deletion of consecutive
nucleobases results in a calibration sequence having at least 80%, at
least 85%, at least 90%, or at least 95% sequence identity with a chosen
standard sequence of a bioagent identifying amplicon.

[0026] In some embodiments, the primers used for amplification of bioagent
identifying amplicons and calibration amplicons hybridize to and amplify
genomic DNA, DNA of bacterial plasmids or DNA of DNA viruses.

[0027] In some embodiments, the primers used for amplification of bioagent
identifying amplicons and corresponding calibration amplicons hybridize
directly to ribosomal RNA or messenger RNA (mRNA) and act as reverse
transcription primers for obtaining DNA from direct amplification of
bacterial rRNA. Methods of amplifying RNA using reverse transcriptase are
well known to those with ordinary skill in the art and can be routinely
established without undue experimentation.

[0028] Synthesis of primers is well known and routine in the art. The
primers may be conveniently and routinely made through the well-known
technique of solid phase synthesis. Equipment for such synthesis is sold
by several vendors including, for example, Applied Biosystems (Foster
City, Calif.). Any other means for such synthesis known in the art may
additionally or alternatively be employed.

[0029] The primers can be employed as compositions for use in methods for
identification of bacterial bioagents as follows: a primer pair
composition is contacted with nucleic acid of an unknown bacterial
bioagent. The nucleic acid is then amplified by a nucleic acid
amplification technique, such as PCR for example, to obtain an
amplification product that represents a bioagent identifying amplicon.
The molecular mass of a single strand or each strand of the
double-stranded amplification product is determined by a molecular mass
measurement technique such as mass spectrometry for example, wherein the
two strands of the double-stranded amplification product are separated
during the ionization process. In some embodiments, the mass spectrometry
is electrospray Fourier transform ion cyclotron resonance mass
spectrometry (ESI-FTICR-MS) or electrospray time of flight mass
spectrometry (ESI-TOF-MS). A list of possible base compositions can be
generated for the molecular mass value obtained for each strand and the
choice of the correct base composition from the list is facilitated by
matching the base composition of one strand with a complementary base
composition of the other strand. The molecular mass or base composition
thus determined is then compared with a database of molecular masses or
base compositions of analogous bioagent identifying amplicons for known
bioagents. A match between the molecular mass or base composition of the
amplification product and the molecular mass or base composition of an
analogous bioagent identifying amplicon for a known bioagent indicates
the identity of the unknown bioagent. In some embodiments, the method is
repeated using a different primer pair to resolve possible ambiguities in
the identification process or to improve the confidence level for the
identification assignment.

[0030] In some embodiments, a bioagent identifying amplicon or a
calibration amplicon may be produced using only a single primer
composition (either the forward or reverse primer of any given primer
pair), provided an appropriate amplification method is chosen, such as,
for example, low stringency single primer PCR (LSSP-PCR).

[0031] In some embodiments, the oligonucleotide primers are "broad range
survey primers" which hybridize to conserved regions of nucleic acid
encoding ribosomal RNA (rRNA) of at least 50%, at least 60%, at least
70%, at least 80%, at least 90%, at least 99%, or all known bacteria and
produce bacterial bioagent identifying amplicons. As used herein, the
term "broad range survey primers" refers to primers that bind to nucleic
acid encoding rRNAs of at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at least 99%, or all known species of bacteria. In
some embodiments, the rRNAs to which the primers hybridize are 16S and
23S rRNAs.

[0032] In some cases, the molecular mass or base composition of a
bacterial bioagent identifying amplicon defined by a broad range survey
primer pair does not provide enough resolution to unambiguously identify
a bacterial bioagent at the species level. These cases benefit from
further analysis of one or more bacterial bioagent identifying amplicons
generated from at least one additional broad range survey primer pair or
from at least one additional "division-wide" primer pair (vide infra).
The employment of more than one bioagent identifying amplicon for
identification of a bioagent is herein referred to as "triangulation
identification" (vide infra).

[0033] In other embodiments, the oligonucleotide primers are
"division-wide" primers which hybridize to nucleic acid encoding genes of
broad divisions of bacteria such as members of the Bacillus/Clostridia
group or members of the α-, β-, γ-, and
ε-proteobacteria. In some embodiments, a division of bacteria
comprises any grouping of bacterial genera with more than one genus
represented. For example, the β-proteobacteria group comprises
members of the following genera: Eikenella, Neisseria, Achromobacter,
Bordetella, Burkholderia, and Raltsonia. Species members of these genera
can be identified using bacterial bioagent identifying amplicons
generated with a primer pair which produces a bacterial bioagent
identifying amplicon from the tufB gene of β-proteobacteria.
Examples of genes to which division-wide primers may hybridize to
include, but are not limited to: RNA polymerase subunits such as rpoB and
rpoC, tRNA synthetases such as valyl-tRNA synthetase (valS) and
aspartyl-tRNA synthetase (aspS), elongation factors such as elongation
factor EF-Tu (tufB), ribosomal proteins such as ribosomal protein L2
(rplB), protein chain initiation factors such as protein chain initiation
factor infB, chaperonins such as groL and dnaK, and cell division
proteins such as peptidase ftsH (hflB).

[0034] In other embodiments, the oligonucleotide primers are designed to
enable the identification of bacteria at the clade group level, which is
a monophyletic taxon referring to a group of organisms which includes the
most recent common ancestor of at least 70%, at least 80%, at least 90%,
or all of its members and at least 70%, at least 80%, at least 90%, or
all of the descendants of that most recent common ancestor. The Bacillus
cereus clade is an example of a bacterial clade group.

[0035] In other embodiments, the oligonucleotide primers are "drill-down"
primers which enable the identification of "sub-species characteristics."
These primers can hybridize to conserved regions of nucleic acid of genes
encoding structural proteins or proteins implicated in, for example,
pathogenicity. Examples of genes indicating sub-species characteristics
include, but are not limited to: toxin genes, pathogenicity markers,
antibiotic resistance genes and virulence factors. Drill down primers
provide the functionality of producing bacterial bioagent identifying
amplicons for drill-down analyses such as strain typing when contacted
with bacterial nucleic acid under amplification conditions.
Identification of such sub-species characteristics is often critical for
determining proper clinical treatment of bacterial infections.

[0036] It is, thus, readily apparent that one with ordinary skill can
design calibration sequences that can be amplified by any of the primer
classes disclosed herein in order to produce appropriate calibration
amplicons.

[0037] One with ordinary skill in the art of design of amplification
primers will recognize that a given primer need not hybridize with 100%
complementarity in order to effectively prime the synthesis of a
complementary nucleic acid strand in an amplification reaction. Moreover,
a primer may hybridize over one or more segments such that intervening or
adjacent segments are not involved in the hybridization event. (e.g: a
loop structure or a hairpin structure). The primers of the present
invention may comprise at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95% or at least 99% sequence identity with
any of the primers listed in Table 1. Thus, in some embodiments of the
present invention, an extent of variation of 70% to 100% of the sequence
identity is possible relative to the specific primer sequences disclosed
herein. Determination of sequence identity is described in the following
example: a primer 20 nucleobases in length which is identical to another
20 nucleobase primer having two non-identical residues has 18 of 20
identical residues (18/20=0.9 or 90% sequence identity). In another
example, a primer 15 nucleobases in length having all residues identical
to a 15 nucleobase segment of primer 20 nucleobases in length would have
15/20=0.75 or 75% sequence identity with the 20 nucleobase primer.

[0038] Percent homology, sequence identity or complementarity, can be
determined by, for example, the Gap program (Wisconsin Sequence Analysis
Package, Version 8 for Unix, Genetics Computer Group, University Research
Park, Madison Wis.), using default settings, which uses the algorithm of
Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some
embodiments, complementarity of primers with respect to the conserved
priming regions of bacterial nucleic acid, is between about 70% and about
80%. In other embodiments, homology, sequence identity or
complementarity, is between about 80% and about 90%. In yet other
embodiments, homology, sequence identity or complementarity, is about
90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 99% or about 100%.

[0039] One with ordinary skill is able to calculate percent sequence
identity or percent sequence homology and able to determine, without
undue experimentation, the effects of variation of primer sequence
identity on the function of the primer in its role in priming synthesis
of a complementary strand of nucleic acid for production of an
amplification product of a corresponding bioagent identifying amplicon.

[0041] In some embodiments, any given primer comprises a modification
comprising the addition of a non-templated T residue to the 5' end of the
primer i.e: the added T residue does not necessarily hybridize to the
nucleic acid being amplified. The addition of a non-templated T residue
has the effect of minimizing the addition of non-templated A residues as
a result of the non-specific enzyme activity of Taq polymerase (Magnuson
et al., Biotechniques, 1996, 21, 700-709), an occurrence which may lead
to ambiguous results arising from molecular mass analysis.

[0042] In some embodiments of the present invention, primers may contain
one or more universal bases. Because any variation (due to codon wobble
in the 3rd position) in the conserved regions among species is
likely to occur in the third position of a DNA triplet, oligonucleotide
primers can be designed such that the nucleotide corresponding to this
position is a base which can bind to more than one nucleotide, referred
to herein as a "universal nucleobase." For example, under this "wobble"
pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and
uridine (U) binds to U or C. Other examples of universal nucleobases
include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et
al., Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degenerate
nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog
containing 5-nitroindazole (Van Aerschot et al., Nucleosides and
Nucleotides, 1995, 14, 1053-1056) or the purine analog
1-(2-deoxy-(3-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al.,
Nucl. Acids Res., 1996, 24, 3302-3306).

[0043] In some embodiments, to compensate for the somewhat weaker binding
by the "wobble" base, the oligonucleotide primers are designed such that
the first and second positions of each triplet are occupied by nucleotide
analogs which bind with greater affinity than the unmodified nucleotide.
Examples of these analogs include, but are not limited to,
2,6-diaminopurine which binds to thymine, 5-propynyluracil which binds to
adenine and 5-propynylcytosine and phenoxazines, including G-clamp, which
binds to G. Propynylated pyrimidines are described in U.S. Pat. Nos.
5,645,985, 5,830,653 and 5,484,908, each of which is commonly owned and
incorporated herein by reference in its entirety. Propynylated primers
are described in U.S. Ser. No. 10/294,203 which is also commonly owned
and incorporated herein by reference in entirety. Phenoxazines are
described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, each of
which is incorporated herein by reference in its entirety. G-clamps are
described in U.S. Pat. Nos. 6,007,992 and 6,028,183, each of which is
incorporated herein by reference in its entirety.

[0044] In some embodiments, non-template primer tags are used to increase
the melting temperature (Tm) of a primer-template duplex in order to
improve amplification efficiency. A non-template tag is designed to
hybridize to at least three consecutive A or T nucleotide residues on a
primer which are complementary to the template. In any given non-template
tag, A can be replaced by C or G and T can also be replaced by C or G.
The extra hydrogen bond in a G-C pair relative to a A-T pair confers
increased stability of the primer-template duplex and improves
amplification efficiency.

[0045] In other embodiments, propynylated tags may be used in a manner
similar to that of the non-template tag, wherein two or more
5-propynylcytidine or 5-propynyluridine residues replace template
matching residues on a primer. In other embodiments, a primer contains a
modified internucleoside linkage such as a phosphorothioate linkage, for
example.

[0046] In some embodiments, the primers contain mass-modifying tags.
Reducing the total number of possible base compositions of a nucleic acid
of specific molecular weight provides a means of avoiding a persistent
source of ambiguity in determination of base composition of amplification
products. Addition of mass-modifying tags to certain nucleobases of a
given primer will result in simplification of de novo determination of
base composition of a given bioagent identifying amplicon (vide infra)
from its molecular mass.

[0047] In some embodiments of the present invention, the mass modified
nucleobase comprises one of the following:
7-deaza-2'-deoxyadenosine-5-triphosphate,
5-iodo-2'-deoxyuridine-5'-triphosphate,
5-bromo-2'-deoxyuridine-5'-triphosphate,
5-bromo-2'-deoxycytidine-5'-triphosphate,
5-iodo-2'-deoxycytidine-5'-triphosphate,
5-hydroxy-2'-deoxyuridine-5'-triphosphate,
4-thiothymidine-5'-triphosphate, 5-aza-2'-deoxyuridine-5'-triphosphate,
5-fluoro-2'-deoxyuridine-5'-triphosphate,
O6-methyl-2'-deoxyguanosine-5'-triphosphate,
N2-methyl-2'-deoxyguanosine-5'-triphosphate,
8-oxo-2'-deoxyguanosine-5'-triphosphate or thiothymidine-5'-triphosphate.
In some embodiments, the mass-modified nucleobase comprises 15N or
13C or both 15N and 13C.

[0048] In some embodiments, bioagent identifying amplicons amenable to
molecular mass determination which are produced by the primers described
herein are either of a length, size or mass compatible with the
particular mode of molecular mass determination or compatible with a
means of providing a predictable fragmentation pattern in order to obtain
predictable fragments of a length compatible with the particular mode of
molecular mass determination. Such means of providing a predictable
fragmentation pattern of an amplification product include, but are not
limited to, cleavage with restriction enzymes or cleavage primers, for
example. Methods of using restriction enzymes and cleavage primers are
well known to those with ordinary skill in the art.

[0049] In some embodiments, amplification products corresponding to
bacterial bioagent identifying amplicons are obtained using the
polymerase chain reaction (PCR) which is a routine method to those with
ordinary skill in the molecular biology arts. Other amplification methods
may be used such as ligase chain reaction (LCR), low-stringency single
primer PCR, and multiple strand displacement amplification (MDA) which
are also well known to those with ordinary skill.

[0050] In the context of this invention, a "bioagent" is any organism,
cell, or virus, living or dead, or a nucleic acid derived from such an
organism, cell or virus. Examples of bioagents include, but are not
limited, to cells, (including but not limited to human clinical samples,
bacterial cells and other pathogens), viruses, fungi, protists,
parasites, and pathogenicity markers (including but not limited to:
pathogenicity islands, antibiotic resistance genes, virulence factors,
toxin genes and other bioregulating compounds). Samples may be alive or
dead or in a vegetative state (for example, vegetative bacteria or
spores) and may be encapsulated or bioengineered. In the context of this
invention, a "pathogen" is a bioagent which causes a disease or disorder.

[0051] In the context of this invention, the term "unknown bioagent" may
mean either: (i) a bioagent whose existence is known (such as the well
known bacterial species Staphylococcus aureus for example) but which is
not known to be in a sample to be analyzed, or (ii) a bioagent whose
existence is not known (for example, the SARS coronavirus was unknown
prior to April 2003). For example, if the method for identification of
coronaviruses disclosed in commonly owned U.S. Ser. No. 10/829,826
(incorporated herein by reference in entirety) was to be employed prior
to April 2003 to identify the SARS coronavirus in a clinical sample, both
meanings of "unknown" bioagent are applicable since the SARS coronavirus
was unknown to science prior to April, 2003 and since it was not known
what bioagent (in this case a coronavirus) was present in the sample. On
the other hand, if the method of U.S. Ser. No. 10/829,826 was to be
employed subsequent to April 2003 to identify the SARS coronavirus in a
clinical sample, only the first meaning (i) of "unknown" bioagent would
apply since the SARS coronavirus became known to science subsequent to
April 2003 and since it was not known what bioagent was present in the
sample.

[0052] In those embodiments wherein the bioagent is an RNA virus, the RNA
of the virus is reverse transcribed to obtain corresponding DNA which can
be subsequently amplified by procedures referred to above. In one
embodiment, one means of reverse transcription is reverse transcriptase,
an enzyme well known in the molecular biology arts.

[0053] The employment of more than one bioagent identifying amplicon for
identification of a bioagent is herein referred to as "triangulation
identification." Triangulation identification is pursued by analyzing a
plurality of bioagent identifying amplicons selected within multiple core
genes. This process can be used to reduce false negative and false
positive signals, and enable reconstruction of the origin of hybrid or
otherwise engineered bioagents. For example, identification of the three
part toxin genes typical of B. anthracis (Bowen et al., J. Appl.
Microbiol., 1999, 87, 270-278) in the absence of the expected signatures
from the B. anthracis genome would suggest a genetic engineering event.

[0054] In some embodiments, the triangulation identification process can
be pursued by characterization of bioagent identifying amplicons in a
massively parallel fashion using the polymerase chain reaction (PCR),
such as multiplex PCR where multiple primers are employed in the same
amplification reaction mixture, or PCR in multi-well plate format wherein
a different and unique pair of primers is used in multiple wells
containing otherwise identical reaction mixtures. Such multiplex and
multi-well PCR methods are well known to those with ordinary skill in the
arts of rapid throughput amplification of nucleic acids.

[0055] In some embodiments, the molecular mass of a given bioagent
identifying amplicon is determined by mass spectrometry. Mass
spectrometry has several advantages, not the least of which is high
bandwidth characterized by the ability to separate (and isolate) many
molecular peaks across a broad range of mass to charge ratio (m/z). Thus
mass spectrometry is intrinsically a parallel detection scheme without
the need for radioactive or fluorescent labels, since every amplification
product is identified by its molecular mass. The current state of the art
in mass spectrometry is such that less than femtomole quantities of
material can be readily analyzed to afford information about the
molecular contents of the sample. An accurate assessment of the molecular
mass of the material can be quickly obtained, irrespective of whether the
molecular weight of the sample is several hundred, or in excess of one
hundred thousand atomic mass units (amu) or Daltons.

[0056] In some embodiments, intact molecular ions are generated from
amplification products using one of a variety of ionization techniques to
convert the sample to gas phase. These ionization methods include, but
are not limited to, electrospray ionization (ES), matrix-assisted laser
desorption ionization (MALDI) and fast atom bombardment (FAB). Upon
ionization, several peaks are observed from one sample due to the
formation of ions with different charges. Averaging the multiple readings
of molecular mass obtained from a single mass spectrum affords an
estimate of molecular mass of the bioagent identifying amplicon.
Electrospray ionization mass spectrometry (ESI-MS) is particularly useful
for very high molecular weight polymers such as proteins and nucleic
acids having molecular weights greater than 10 kDa, since it yields a
distribution of multiply-charged molecules of the sample without causing
a significant amount of fragmentation.

[0057] The mass detectors used in the methods of the present invention
include, but are not limited to, Fourier transform ion cyclotron
resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic
sector, time of flight (TOF), Q-TOF, and triple quadrupole.

[0058] In some embodiments, conversion of molecular mass data to a base
composition is useful for certain analyses. As used herein, a "base
composition" is the exact number of each nucleobase (A, T, C and G). For
example, amplification of nucleic acid of Neisseria meningitidis with a
primer pair that produces an amplification product from nucleic acid of
23S rRNA that has a molecular mass (sense strand) of 28480.75124, from
which a base composition of A25 G27 C22 T18 is assigned from a list of
possible base compositions calculated from the molecular mass using
standard known molecular masses of each of the four nucleobases.

[0059] In some embodiments, assignment of base compositions to
experimentally determined molecular masses is accomplished using "base
composition probability clouds." Base compositions, like sequences, vary
slightly from isolate to isolate within species. It is possible to manage
this diversity by building "base composition probability clouds" around
the composition constraints for each species. This permits identification
of organisms in a fashion similar to sequence analysis. Optimal primer
design requires optimal choice of bioagent identifying amplicons and
maximizes the separation between the base composition signatures of
individual bioagents. Areas where clouds overlap indicate regions that
may result in a misclassification, a problem which is overcome by a
triangulation identification process using bioagent identifying amplicons
not affected by overlap of base composition probability clouds.

[0060] In some embodiments, base composition probability clouds provide
the means for screening potential primer pairs in order to avoid
potential misclassifications of base compositions. In other embodiments,
base composition probability clouds provide the means for predicting the
identity of a bioagent whose assigned base composition was not previously
observed and/or indexed in a bioagent identifying amplicon base
composition database due to evolutionary transitions in its nucleic acid
sequence. Thus, in contrast to probe-based techniques, mass spectrometry
determination of base composition does not require prior knowledge of the
composition or sequence in order to make the measurement.

[0061] The present invention provides bioagent classifying information
similar to DNA sequencing and phylogenetic analysis at a level sufficient
to detect and identify a given bioagent. Furthermore, the process of
determination of a previously unknown base composition for a given
bioagent (for example, in a case where sequence information is
unavailable) has downstream utility by providing additional bioagent
indexing information with which to populate base composition databases.
The process of future bioagent identification is thus greatly improved as
more BCS indexes become available in base composition databases.

[0062] The present invention also provides kits for carrying out the
methods described herein. In some embodiments, the kit may comprise a
sufficient quantity of one or more primer pairs to perform an
amplification reaction on a target polynucleotide from a bioagent to form
a bioagent identifying amplicon. In some embodiments, the kit may
comprise from one to fifty primer pairs, from one to twenty primer pairs,
from one to ten primer pairs, or from two to five primer pairs. In some
embodiments, the kit may comprise one or more primer pairs recited in
Table 1.

[0063] In some embodiments, the kit may comprise broad range survey
primers, division wide primers, clade group primers or drill-down
primers, or any combination thereof. A kit may be designed so as to
comprise particular primer pairs for identification of a particular
bioagent. For example, a broad range survey primer kit may be used
initially to identify an unknown bioagent as a member of the
Bacillus/Clostridia group. Another example of a division-wide kit may be
used to distinguish Bacillus anthracis, Bacillus cereus and Bacillus
thuringiensis from each other. A drill-down kit may be used, for example,
to identify genetically engineered Bacillus anthracis. In some
embodiments, any of these kits may be combined to comprise a combination
of broad range survey primers and division-wide primers so as to be able
to identify the species of an unknown bioagent.

[0064] In some embodiments, the kit may contain standardized nucleic acids
for use as internal amplification calibrants.

[0065] In some embodiments, the kit may also comprise a sufficient
quantity of reverse transcriptase (if an RNA virus is to be identified
for example), a DNA polymerase, suitable nucleoside triphosphates
(including any of those described above), a DNA ligase, and/or reaction
buffer, or any combination thereof, for the amplification processes
described above. A kit may further include instructions pertinent for the
particular embodiment of the kit, such instructions describing the primer
pairs and amplification conditions for operation of the method. A kit may
also comprise amplification reaction containers such as microcentrifuge
tubes and the like. A kit may also comprise reagents or other materials
for isolating bioagent nucleic acid or bioagent identifying amplicons
from amplification, including, for example, detergents, solvents, or ion
exchange resins which may be linked to magnetic beads. A kit may also
comprise a table of measured or calculated molecular masses and/or base
compositions of bioagents using the primer pairs of the kit.

[0066] While the present invention has been described with specificity in
accordance with certain of its embodiments, the following examples serve
only to illustrate the invention and are not intended to limit the same.
Throughout these examples, molecular cloning reactions, and other
standard recombinant DNA techniques, may be carried out according to
methods described in Maniatis et al., Molecular Cloning--A Laboratory
Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially
available reagents, except where otherwise noted.

[0067] This example describes the design of two coronavirus calibrant
polynucleotides based on viral bioagent identifying amplicons for
identification of coronaviruses (viral bioagent identifying amplicons) in
the RNA-dependent RNA polymerase (RdRp) gene and in the nsp11 gene which
are described in a method for identification of coronaviruses disclosed
in U.S. application Ser. No. 10/829,826. The primers used to define the
viral bioagent identifying amplicons hybridize to regions of the RdRp
gene (primer pair no. 453:
forward--TAAGUaUaTUaATGGCGGCUaGG (SEQ ID NO: 1) and
reverse--TTTAGGATAGTCaCaCa AACCCAT (SEQ ID NO: 2)) and the
nsp11 gene (primer pair no. 455: forward--TGTTTG
UaUaUaUaGGAATTGTAATGTTGA (SEQ ID NO: 3) and
reverse--TGGAATGCATGCUa UaAUaUaAACATACA (SEQ ID NO:
4)), wherein Ua represents=5-propynyluracil and Ca represents
5-propynylcytosine). The calibration sequence chosen to simulate the RdRp
calibration amplicon is SEQ ID NO: 5 which corresponds to positions 15146
to 15233 of NC--004718.3 (SARS coronavirus TOR2 genome) with
deletion of positions 15179-15183 to yield a calibration amplicon length
of 83 bp. The calibration sequence for the nsp11 calibration amplicon is
SEQ ID NO: 6, which corresponds to positions 19113 to 19249 of
NC--004718.3 (SARS coronavirus TOR2 genome) with deletion of
positions 19172-19176 to yield a calibration amplicon of 132 bp length.
Both calibrant standard sequences (SEQ ID NOs: 5 and 6) were included on
a single polynucleotide (SEQ ID NO: 7--herein designated a "combination
calibration polynucleotide") which was cloned into a pCR®-Blunt
vector (Invitrogen, Carlsbad, Calif.). Thus, when the combination
calibration polynucleotide is added to an amplification reaction, an
RdRp-based calibration amplicon will be produced in an amplification
reaction with primer pair 453 (SEQ ID NOs: 1:2) and an nsp11-based
calibration amplicon will be produced with primer pair 455 (SEQ ID NOs:
3:4).

[0068] The viral bioagent identifying amplicons are used as identifiers of
coronaviruses due to the variable regions between the conserved priming
regions which can be distinguished by mass spectrometry. The calibration
polynucleotides are used to produce calibration amplicons from which the
quantity of identified coronavirus is determined.

Example 2

Use of a Calibration Polynucleotide for Determining the Quantity of
Coronavirus in a Clinical Sample

[0069] To determine the quantity of SARS coronavirus in a clinical sample,
viable SARS coronavirus was added to human serum and analyzed. The TOR2
isolate of the SARS coronavirus from three passages in Vero cells was
titered by plaque assay. Virus was handled in a P3 facility by
investigators wearing forced air respirators. Equipment and supplies were
decontaminated with 10% hypochlorite bleach solution for a minimum of 30
minutes or by immersion in 10% formalin for a minimum of 12 hours and
virus was handled in strict accordance with specific Scripps Research
Institute policy. SARS coronavirus was cultured in sub confluent Vero-E6
cells at 37° C., 5% CO2 in complete DMEM with final
concentrations of 10% fetal bovine serum (Hyclone, Salt Lake City, Utah),
292 μg/mL L-Glutamine, 100 U/mL penicillin G sodium, 100 μg/mL
streptomycin sulfate (Invitrogen, Carlsbad Calif.), and 10 mM HEPES
(Invitrogen, Carlsbad Calif.). Virus-containing medium was collected
during the peak of viral cytopathic effects, 48 h after inoculation with
approximately 10 PFU/cell of SARS coronavirus from the second passage of
stock virus. Infectious virus was titered by plaque assay. Monolayers of
Vero-E6 cells were prepared at 70-80% confluence in tissue culture
plates. Serial tenfold dilutions of virus were prepared in complete DMEM.
Medium was aspirated from cells, replaced by 200 μL of inoculum, and
cells were incubated at 37° C., 5% CO2 for 1 hour. Cells were
overlaid with 2-3 mL/well of 0.7% agarose, lx DMEM overlay containing 2%
fetal bovine serum. Agarose was allowed to solidify at room temperature
then cells were incubated at 37° C., 5% CO2 for 72 h. Plates
were decontaminated by overnight formalin immersion, agarose plugs were
removed, and cells were stained with 0.1% crystal violet to highlight
viral plaques.

[0070] RNA was isolated from serum containing two different concentrations
of the virus (1.7×105 and 170 PFU/mL) and reverse transcribed
to cDNA using random primers and reverse transcriptase. A PFU (plate
forming unit) is a quantitative measure of the number of infectious virus
particles in a given sample, since each infectious virus particle can
give rise to a single clear plaque on infection of a continuous "lawn" of
bacteria or a continuous sheet of cultured cells. PCR amplifications were
performed using both the RdRp and the nsp11 primer sets on serial
ten-fold dilutions of these cDNAs. Amplification products were purified
and analyzed by methods commonly owned and disclosed in U.S. application
Ser. Nos. 10/829,826 and 10/844,938, each of which is incorporated herein
by reference in its entirety. The limit of SARS coronavirus detection was
10-2 PFU per PCR reaction (˜1.7 PFU/mL serum). Since PFU reflects
the number of infectious viral particles and not the total number of RNA
genomes, the number of reverse-transcribed SARS genomes was estimated by
competitive, quantitative PCR using a calibration polynucleotide.
Analysis of ratios of mass spectral peak heights of titrations of the
calibration polynucleotide and the SARS cDNA showed that approximately
300 reverse-transcribed viral genomes were present per PFU, similar to
the ratio of viral genome copies per PFU reported for RNA viruses (J. S.
Towner et al., J Virol In Press (2004)). Using this estimate, the PCR
primers were sensitive to three genomes per PCR reaction, consistent with
previously reported detection limits for optimized SARS-specific primers
(Drosten et al., New England Journal of Medicine, 2003, 348, 1967). When
RT-PCR products were measured for varying dilutions of the SARS virus
spiked directly into serum, 1 PFU (˜300 genomes) per PCR reaction
or 170 PFU (5.1×104 genomes) per mL serum could be reliably
detected. The discrepancy between the detection sensitivities in the two
experimental protocols described above suggests that there were losses
associated with RNA extraction and reverse transcription when very little
virus was present (<300 copies) in the starting sample in serum.

[0071] To determine the relationship between PFU and copies of nucleic
acid target, the virus stock was analyzed using the methods of the
present invention. Synthetic DNA templates with nucleic acid sequence
identical in all respects to RdRp-based (SEQ ID NO: 5) and nsp11-based
(SEQ ID NO: 6) viral bioagent identifying amplicons for the SARS
coronavirus with the exception of 5 base deletions internal to each
amplicon were combined into a single combination calibration
polynucleotide (SEQ ID NO: 7) and cloned into a pCR®-Blunt vector
(Invitrogen, Carlsbad, Calif.) to produce a calibration polynucleotide.
The calibrant plasmid stock solutions were quantified using OD260
measurements, serially diluted (10-fold dilutions), and mixed with a
fixed amount of post-reverse transcriptase cDNA preparation of the virus
stock and analyzed by competitive PCR and electrospray mass spectrometry.
Each PCR reaction produced two sets of amplicons, one corresponding to
the calibrant amplicon and the other to the viral bioagent identifying
amplicon. Since the primers hybridize to both the calibration
polynucleotide and the coronavirus cDNA, it was reasonably assumed that
the calibration polynucleotide and coronavirus cDNA would have similar
PCR efficiencies for amplification of the two products. Analysis of the
ratios of peak heights (abundance data) of the resultant mass spectra of
the calibration amplicons DNA and viral bioagent identifying amplicons
used to determine the amounts of nucleic acid copies (as measured by
calibrant molecules) present per PFU. Since all of the extracted RNA was
used in the reverse transcriptase step to produce the viral cDNA, the
approximate amount of nucleic acids associated with infectious virus
particles in the original viral preparation could be estimated. Mass
spectrometry analysis showed an approximate 1:1 peak abundance between
the calibrant peak at the 3×104 copy number dilution and the
viral bioagent identifying amplicon peak for the RdRp primer set (FIG.
2). Thus, the relationship between PFU and copies of nucleic acid was
calculated to be 1 PFU=300 copies of nucleic acid.

[0072] The calibration sequences described in this example are appropriate
for use in production of calibration amplicons which are in turn useful
for determining the quantity of all known members of the coronavirus
family. Further, it is reasonably expected that these calibration
sequences will likewise be appropriate for quantification of any
coronaviruses that are yet to be discovered.

Example 3

Design of Calibrant Polynucleotides Based on Bioagent Identifying
Amplicons for Identification of Species of Bacteria (Bacterial Bioagent
Identifying Amplicons)

[0073] This example describes the design of 19 calibrant polynucleotides
based on broad range bacterial bioagent identifying amplicons. The
bacterial bioagent identifying amplicons are obtained upon amplification
of bacterial nucleic acid with primers (Table 1) that have been disclosed
in U.S. patent application Ser. Nos. 10/660,122, 10,728,486, and
60/559,754, each of which is commonly owned and incorporated herein by
reference in its entirety.

[0074] Calibration sequences were designed to simulate bacterial bioagent
identifying amplicons produced by the primer pairs shown in Table 1. The
calibration sequences were chosen as a representative member of the
section of bacterial genome from specific bacterial species which would
be amplified by a given primer pair. The model bacterial species upon
which the calibration sequences are based are also shown in Table 1. For
example, the calibration sequence chosen to correspond to an amplicon
produced by primer pair no. 346 is SEQ ID NO: 8. In Table 1, the forward
(_F) or reverse (_R) primer name indicates the coordinates of an
extraction representing a gene of a standard reference bacterial genome
to which the primer hybridizes e.g.: the forward primer name
16S_EC--713--732TMOD_F indicates that the forward primer
hybridizes to residues 712-732 of the gene encoding 16S ribosomal RNA in
an E. coli reference sequence (in this case, the reference sequence (SEQ
ID NO: 66 in Table 2) is an extraction consisting of residues
4033120-4034661 of the genomic sequence of E. coli K12 (GenBank Accession
No. NC--000913)--See Table 2. Additional gene coordinate reference
information is shown in Table 2. The designation "TMOD" in the primer
names indicates that the 5' end of the primer has been modified with a
non-matched template T residue. This modification prevents the PCR
polymerase from adding non-templated adenosine residues to the 5' end of
the amplification product, an occurrence which may result in
miscalculation of base composition from molecular mass data.

[0075] The 19 calibration sequences shown in Table 1 were combined into a
single calibration polynucleotide sequence (SEQ ID NO: 9--which is herein
designated a "combination calibration polynucleotide") which was then
cloned into a pCR®-Blunt vector (Invitrogen, Carlsbad, Calif.). This
combination calibration polynucleotide can be used in conjunction with
the primers of Table 1 as an internal standard to produce calibration
amplicons for use in determination of the quantity of any bacterial
bioagent. Thus, for example, when the combination calibration
polynucleotide vector is present in an amplification reaction mixture, a
calibration amplicon based on primer pair 346 (16S rRNA) will be produced
in an amplification reaction with primer pair 346 and a calibration
amplicon based on primer pair 363 (rpoC) will be produced with primer
pair 363.

Use of a Calibration Polynucleotide for Determining the Quantity of
Bacillus Anthracis in a Sample Containing a Mixture of Microbes

[0076] The capC gene is a gene involved in capsule synthesis which resides
on the pX02 plasmid of Bacillus anthracis. Primer pair no. 350 (see
Tables 1 and 2) was designed to identify Bacillus anthracis via
production of a bacterial bioagent identifying amplicon. Known quantities
of the combination calibration polynucleotide vector described in Example
3 were added to amplification mixtures containing bacterial bioagent
nucleic acid from a mixture of microbes which included the Ames strain of
Bacillus anthracis. Upon amplification of the bacterial bioagent nucleic
acid and the combination calibration polynucleotide vector with primer
pair no. 350, bacterial bioagent identifying amplicons and calibration
amplicons were obtained and characterized by mass spectrometry. A
spectrum of an amplified nucleic acid mixture containing the Ames strain
of Bacillus anthracis, a known quantity of combination calibration
polynucleotide vector which includes the CapC calibration sequence for
Bacillus anthracis and primer pair 350 is shown in FIG. 3. The molecular
masses of the bioagent identifying amplicons provided the means for
identification of the bioagent from which they were obtained (Ames strain
of Bacillus anthracis) and the molecular masses of the calibration
amplicons provided the means for their identification as well. The
relationship between the abundance (peak height) of the calibration
amplicon signals and the bacterial bioagent identifying amplicon signals
provides the means of calculation of the copies of the pX02 plasmid of
the Ames strain of Bacillus anthracis. Methods of calculating quantities
of molecules based on internal calibration procedures are well known to
those of ordinary skill in the art.

[0077] Calibration amplicons and bacterial bioagent identifying amplicons
produced in the reaction are visible in the mass spectrum as indicated
and abundance (peak height) data are used to calculate the quantity of
the pX02 plasmid of the Ames strain of Bacillus anthracis in the sample.
Averaging the results of 10 repetitions of the experiment described
above, enabled a calculation that indicated that the quantity of Ames
strain of Bacillus anthracis present in the sample corresponds to
approximately 10 copies of pX02 plasmid.

[0078] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from the
foregoing description. Such modifications are also intended to fall
within the scope of the appended claims. Each reference (including, but
not limited to, journal articles, U.S. and non-U.S. patents, patent
application publications, international patent application publications,
gene bank accession numbers, and the like) cited in the present
application is incorporated herein by reference in its entirety.